Electrochemical detection of polymerase reactions by specific metal-phosphate complex formation

ABSTRACT

The present invention relates to determining pyrophosphate by electrochemical means via the depletion of metal ions as a result of its binding to and/or precipitation with pyrophosphate. This principle is of particular interest for polymerase catalysed reactions such as nucleic acid sequencing.

FIELD OF THE INVENTION

The present invention is in the field of chemistry and biology.Specifically, the invention relates to the electrochemical detection ofpyrophosphate and to electrochemical nucleic acid sequencing.

INTRODUCTION

Nucleic acid polymerase reactions are central to a multitude ofsensitive detection methods. Especially many different solutions havebeen developed for PCR product formation. More specifically, a certainreaction used to identify a DNA sequence termed pyrosequencing (Ahmadianet al. Clin. Chim. Acta. 2006. 363(1-2):83-94) is of great interest. Inpyrosequencing the four different nucleotides are added to a polymerasereaction in alternating cycles and the release of pyrophosphate ismonitored indirectly.

Currently, two main approaches for the detection of product formationfrom a polymerase reaction such as a nucleic acid sequencing methodexist:

Pyrophosphate is used to generate ATP via sulfurylase and ADPS, whichleads to light emission by luciferase (Ahmadian et al. Clin. Chim. Acta.2006. 363(1-2):83-94). The light is detected and corresponds to a givenreaction by polymerase. As a disadvantage, this method utilizesexpensive and unstable agents, such as enzymes and ATP-derivates.

Another method is employed by Ion Torrent that measures the polymerasereaction by a change in pH (Rothberg et al. Nature. 2011 Jul. 21;475(7356):348-52). The reaction liberates one extra H⁺-ion which isdetected by a chemFET or ISFET (see WO2010047804). The advantage of thismethod is a simple adaptation for integrated circuits which allowsconsiderable miniaturization and parallelization. No extra enzymes otherthan the polymerase are necessary. However, the reaction needs to occurlargely unbuffered and suffers from a low sensitivity.

In other methods pyrophosphate is detected by complexation with certainmetal ions. Calcein/Mn²⁺ is a colourless complex, which becomesfluorescent when the manganese is depleted and occupied with magnesium(Tomita et al. Nat Protoc. 2008; 3(5):877-82.). Alternatively,pyrophosphate has been used to measure manganese ions in solution byoptical means (Takashima et al. J. American Chem. Soc. [Internet]. 2011Dec. 28).

Aluminium ions also form a tight specific complex with pyrophosphate. Afluorescent rhodamine/Al³⁺ complex is depleted of aluminium by thesynthesis of pyrophosphate and thus becomes colorless for detection(Lohani et al. Analyst. 2010. 135(8):2079-84). Free, uncomplexed AI(III)can be detected with ISFET (Abbaspour et al. Anal. Chim. Acta. 2010 Mar.3; 662(1):76-81.). A chelator-modified silicon-on-insulator field-effecttransistor (SOI-FET) with bound Zn²⁺ ions has been shown to detectpyrophosphate liberated by a polymerase reaction directly by theimmobilized complex (Credo et al. The Analyst [Internet]. 2012 Jan. 19).

Several other metal ions are known to be specifically displaced fromcomplexes (e.g. transferrin) by pyrophosphate, e.g. Fe³⁺, Ga³⁺, In³⁺(Harris et al. Coordination Chemistry Reviews. 2002. 228(2):237-62),Sn²⁺ and Zn²⁺ (Cigala et al. Journal of Molecular Liquids. 2012.165:143-153).

Some lanthanide metal ions such as terbium, europium and ytterbium areknown to bind to phosphate species such as triphosphates, DNA,pyrophosphate, and phosphate, albeit with different affinities (Spangleret al. Ann. N. Y. Acad. Sci. 2008. 1130:138-48.). These metal ions canbe used either free or in complex with certain ligands that may increasetheir specificity. So far, these measurements were conducted by usingthe unique fluorescence properties of lanthanides.

The main approaches suffer from the drawback that they either requireexpensive and unstable agents, or the reaction is carried out in alargely unbuffered system and has a low sensitivity. Methods based onthe formation of metal ion/pyrophosphate complexes require extrahardware such as optical devices or special surface receptors (for themethod of Credo et al.) which is expensive and hard to implement intoexisting sequencing units.

Recently, synthetic nucleotide analogues lacking terminal pyrophosphatemoieties were successfully tested in polymerase reactions (Herdewijn andMarlière. FEBS Lett. 2012. Jul. 16; 586(15):2049-56.). The pyrophosphateleaving group was replaced by synthetic compounds that were equallyliberated upon base incorporation by various polymerases. Since many ofthe effective leaving groups are metal ion chelating groups such asaspartic acid, iminodipropionic acid, or iminodiacetic acid, it isconceivable that the detection principle of polymerase reactions bymetal ion binding leaving groups using such nucleotide analogues remainsthe same.

The problem to be solved by the present invention may be formulated asthe provision of an inexpensive and simple method for a fast andsensitive detection of nucleic acid polymerase activity with a potentialfor miniaturization and parallelization. Ideally, the method may beintegrated into current systems such as sequencing devices without orwith few modifications.

DEFINITIONS Sample

A sample refers to any kind of chemical or biological substance orsubstance mixture comprising a nucleic acid, wherein the sequence orpart of the sequence of the nucleic acid is of interest. A sample maystem from or comprise a prokaryote or an eukaryote, such as an archaeon,a bacterium, a protist, a fungus, a virus, a viroid, a plant, an animalor a synthetic oligonucleotide.

Nucleic Acid

The term (template) nucleic acid is here used in its broadest sense andcomprises ribonucleic acids (RNA) and deoxyribonucleic acids (DNA) fromall possible sources, in all lengths and configurations, such asdouble-stranded, single-stranded, circular, linear or branched. Allsub-units and sub-types are also comprised, such as monomericnucleotides, oligomers, plasmids, viral and bacterial nucleic acids, aswell as genomic and non-genomic DNA and RNA from animal and plant cellsor other eukaryotes or prokaryotes, mRNA (messenger RNA) in processedand unprocessed form, tRNA (transfer RNA), hn-RNA (heterogeneous nuclearRNA), rRNA (ribosomal RNA), LNA (locked nucleic acid), mtRNA(mitochondrial), nRNA (nuclear RNA), siRNA (short interfering RNA),snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNA (SmallCajal Body specific RNA), microRNA, dsRNA (double-stranded RNA),ribozyme, riboswitch, viral RNA, dsDNA (double-stranded DNA), ssDNA(single-stranded DNA), plasmid DNA, cosmid DNA, chromosomal DNA, viralDNA, mtDNA (mitochondrial DNA), nDNA (nuclear DNA), snRNA (small nuclearDNA) or the like or as well as all other conceivable nucleic acids.

Sometimes the concentration of the nucleic acid to be sequenced(template nucleic acid) may be too low for sequencing. In this case, thesample may be subjected to an amplification method prior to sequencing.Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including but notlimited to the polymerase chain reaction (PCR), ligase chain reaction(LCR), transcription-based amplification system (TAS), nucleic acidsequence based amplification (NASBA), rolling circle amplification(RCA), transcription-mediated amplification (TMA), self-sustainingsequence replication (3SR) and Qβ amplification. In a preferredembodiment the amplification method is selected from the group ofpolymerase chain reaction (PCR), real-time PCR (rtPCR),helicase-dependent amplification (HDA) and recombinase-polymeraseamplification (RPA).

Primer

The sequence of the primer (molecule) may be a random sequence. However,in cases where part of the sequence of the template nucleic acid isalready known the primer can be designed to be complementary to such asequence. In one embodiment, the primer anneals to either the 3′ or the5′ end of the template nucleic acid. The primer may be prepared usingany suitable method, such as, for example, the phosphotriester andphosphodiester methods or automated embodiments thereof. In one suchautomated embodiment diethylophosphoramidites are used as startingmaterials and may be synthesized as described by Beaucage et al.Tetrahedron Letters. 1981. 22:1859-1862. One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,006, which is hereby incorporated by reference. It is alsopossible to use a primer which has been isolated from a biologicalsource (such as a restriction endonuclease digest).

Preferred primers have a length of about 15-100, more preferably about20-50, most preferably about 20-40 bases.

Nucleotides

As used herein, the term nucleotides refer to deoxyribonucleosidepolyphosphates, such as deoxyribonucleoside triphosphates (dNTPs).Non-limiting examples of such dNTPs are dATP, dGTP, dCTP, dTTP, dUTP,which may also be present in the form of labelled derivatives, forinstance comprising a fluorescent label, a radioactive label, a biotinlabel. dNTPs with modified nucleotide bases are also encompassed,wherein the nucleotide bases are for example hypoxanthine, xanthine,7-methylguanine, inosine, xanthinosine, 7-methylguanosine,5,6-dihydrouracil, 5-methylcytosine, pseudouridine, dihydrouridine,5-methylcytidine. Deoxyribonucleoside polyphosphates, i.e. nucleotideswith more than 3 phosphates, are also utilized by polymerases. In thiscase the polymerase reaction does not generate pyrophosphate.

However, the principle of selecting a metal ion for binding to otherphosphates (such as triphosphate) and using it for electrochemicaldetecting said phosphate can be applied correspondingly.

Elongation

Nucleic acid elongation herein means the stepwise addition ofnucleotides to the growing nucleic acid chain. Elongation is catalyzedby a polymerase. Herein, a polymerase include but are not limited to T7DNA polymerase, DNA Polymerase y, Escherichia coli DNA pol I, Thermusaquaticus pol I, Bacillus stearothermophilus pol I, Pol II (bacterial),Phi29 DNA polymerase, Pol B (archaebacterial), Pol α, δ, ε and ζ,eukaryotic polymerase pol β, pol σ, pol λ, pol μ, terminaldeoxynucleotidyltransferase (TdT), Pol X polymerase and Pol IV.

Sequencing

Nucleic acid sequencing can consist of determining whether a particularnucleic acid differs in sequence from a reference nucleic acid,confirming the presence of a particular nucleic acid sequence in asample, determining partial sequence information such as the identity ofone or more nucleotides within a nucleic acid, determining the identityand order of nucleotides within a nucleic acid, etc.

Most sequencing methods relate to the sequencing of DNA. Currentmethods, thus, typically require RNA to be converted to complementaryDNA (cDNA) via reverse transcription prior to sequencing. Thus, if RNAis to be sequenced, the RNA may be first reverse transcribed into cDNA.

Annealing

Annealing refers to the pairing of the primer by hydrogen bonds to acomplementary sequence on the template nucleic acid, forming adouble-stranded polynucleotide. Annealing may be facilitated bydecreasing the temperature.

Electrode Measurements

Electrochemical measurements may be made in an electrochemical cellconsisting of two or more electrodes and the electronic circuitry forcontrolling and measuring the current and the potential.

The simplest electrochemical cell uses two electrodes. The potential ofone electrode is sensitive to the analyte's concentration, and is calledthe working electrode or the indicator electrode, herein only electrode.The second electrode, which is called reference electrode, completes theelectrical circuit and provides a reference potential against which theworking electrode's potential is measured. Ideally the referenceelectrode's potential remains constant so that one can assign to theworking electrode any change in the overall cell potential.

Potentiometry is the field of electroanalytical chemistry in whichpotential is measured under the conditions of no current flow. Themeasured potential may then be used to determine the analytical quantityof interest, generally the concentration of some component of theanalyte solution. The potential that develops in the electrochemicalcell is the result of the free energy change that would occur if thechemical phenomena were to proceed until the equilibrium condition hasbeen satisfied.

A reference electrode is an electrode which has a stable and well-knownelectrode potential. Reference electrodes are well-known to the skilledperson.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have unexpectedly found that biological reactions whichrelease pyrophosphate may be detected by electrochemical methods via theformation of pyrophosphate/metal ion complexes and/or precipitates.

The present invention is based on the principle that pyrophosphate (PPi;inorganic diphosphate) can be bound and/or precipitated directly in abiological reaction (in a reaction mixture). The binding and/orprecipitation decreases the concentration of unbound and/or dissolved(free) PPi which changes the concentration of a metal ion, subsequentlyresulting in a change of signal generation in an electrochemical sensor.

A typical PPi releasing reaction is a reaction that uses the syntheticactivity of a polymerase. For example, DNA polymerase catalyses thepolymerization of deoxyribonucleotides (dNTPs) into a DNA strand. Theaddition of one of the four complementary dNTPs onto the templatereleases PPi stoichiometrically. This principle can be exploited in thatthe synthetic activity of the polymerase is determined indirectly overthe release of PPi. In contrast to the present methods, the depletion ofmetal ions as a result of the binding to and/or the precipitation withPPi is detected by electrochemical means. Metal ions that bind to thepyrophosphate anion specifically in the presence of triphosphates andnucleic acids can thereby be used as an indicator for polymerasereactions. Free metal ions can be detected by electrochemical methodsand thus indirectly indicate the amount of pyrophosphate complex formedby the reaction despite the presence of triphosphates andoligonucleotides. The decrease in free metal ion concentration resultsin a corresponding signal drop at an electrode. This will occur only ifa nucleotide matching to the template is offered to the polymerase.Thus, this technique can be applied to the detection of nucleic acidsequencing reactions.

Sequencing

Accordingly, in a first aspect the present invention relates to anelectrochemical nucleic acid sequencing method comprising the steps of(a) providing a sample comprising a template nucleic acid to besequenced; (b) annealing a primer molecule to said template nucleic acidor introducing a nick at a predetermined site into a double strandedtemplate; (c) carrying out nucleic acid elongation steps with apolymerase on said template in the presence of metal ions, wherein eachstep comprises the addition of a single type of nucleotide, whereinpyrophosphate is released in a stoichiometric ratio to the number ofnucleotides incorporated, and wherein the metal ions are capable ofspecifically binding to pyrophosphate; (d) determining in said samplethe change of potential, current or charge at an electrode and (e)correlating this change in potential, current or charge over the numberof elongation steps with the type of nucleotides added.

To correlate the signal observed with the sequence of the templatenucleic acid to be sequenced one may proceed as follows:

A change in the signal may be observed, once a nucleotide issuccessfully incorporated. Since only one type of nucleotide (adenine A,cytosine C, guanine G, or uracil U/thymine T) is added at the same time,one can deduce the type of incorporated (elongated) nucleotide.Repeating the steps of adding types of nucleotides and observing thesignal one may further deduce the whole sequence of the template nucleicacid.

Preferably, a calibration curve is generated by measuring the signal atvarious known concentrations of metal ions used for binding and/orprecipitating pyrophosphate in order to ascertain confidence parameters.For example pyrophosphate may be added in various known concentrationsto a solution of constant metal ion concentration in another experiment.The calibration curve is preferably made in a solution (e.g. a buffer)which is comparable to the test solution (e.g. sequencing buffer). Inthe test solution the initial metal ion concentration may be known orcalculated from the initial signal.

Preferably, the nucleic acid sequencing method is based onpyrosequencing, reversible-terminator-pyrosequencing, or closed complexformation sequencing.

It is preferred that step d. and optionally step e. is done after eachelongation step c. Alternatively, step d. and optionally step e. is doneafter every second, third, fourth, firth or even more elongation stepsc.

The sequencing method according to the invention is preferably based onpyrosequencing. Pyrosequencing is based on the detection of thepyrophosphate (PPi) that is released during DNA polymerization (see,e.g., U.S. Pat. Nos. 6,210,891 and 6,258,568). While avoiding the needfor electrophoretic separation, pyrosequencing suffers from a largenumber of drawbacks that have as yet limited its widespreadapplicability (Franca et al. Quarterly Reviews of Biophysics. 2002.35(2):169-200).

In an alternative embodiment the step of sequencing involvesreversible-terminator-pyrosequencing. The method is extensivelyexplained in Wu et al. PNAS. 2007. 104(42):16462-16467, which is herebyincorporated by reference.

In yet another embodiment sequencing involves closed complex formationsequencing. The method is described in WO 2007/048033, which is herebyincorporated by reference.

Polymerase Reaction

The inventive concept is particularly advantageous in the case ofsequencing a nucleic acid. There are, however, other cases where it isdesirable to detect and/or quantify the amount (e.g. concentration) or achange in said amount of PPi with a fast and cost-effective method.Consequently, the invention relates to a method for detecting a nucleicacid polymerase reaction comprising the steps of (a) providing a samplecomprising a nucleic acid; (b) carrying out a nucleic acid polymerasereaction step on said sample in the presence of metal ions, whereinpyrophosphate is released or depleted depending on the type ofpolymerase reaction, and wherein the metal ions are capable ofspecifically binding to pyrophosphate; (c) determining the potential,current or charge at an electrode at least twice, wherein between thefirst time and the second time one or more nucleic acid polymerasereaction steps take place; and (d) correlating the difference inpotential, current or charge over the number of polymerase reactionsteps and/or time with the progress of the nucleic acid polymerasereaction.

It is preferred that the nucleic acid polymerase reaction is a synthesisreaction, an amplification reaction and/or a transcription reaction.

It is further preferred that the step of determining the potential,current or charge (step c.) and optionally the step of correlating thedifference in potential, current or charge step (step d.) is done aftereach polymerase reaction step (step b.). Alternatively, step c. andoptionally step d. is done after every second, third, fourth, fifth oreven more elongation polymerase reaction steps.

As described above, the principle can generally be utilized forassessing whether the concentration of pyrophosphate changes in asample. The method of detecting the formation or depletion ofpyrophosphate in a sample comprises the following steps: (a) providing asample in which pyrophosphate is formed or depleted; (b) adding to saidsample metal ions, wherein the metal ions are capable of preferentiallybinding pyrophosphate; (c) determining the potential, current or chargein the sample a first and at least a second time; (d) correlating thedifference in potential, current or charge over time with the formationor depletion of pyrophosphate in the sample.

Further, the invention can further be used for detecting the presenceand/or the quantity, e.g. concentration, of pyrophosphate in a sample.This method comprises the steps of (a) adding to a sample comprisingpyrophosphate a predetermined amount of metal ions or metal ions of aknown potential, current or charge, wherein the metal ions are capableof specifically binding pyrophosphate; (b) determining in said samplethe potential, current or charge at an electrode; and (c) correlatingthe potential, current or charge with the amount of pyrophosphatepresent in said sample.

Metal Ions

There are a number of different metal ions that can bind to and/orprecipitate pyrophosphate and, thus, be used in the present method.Preferred metal ions are manganese ions, tin ions, zinc ions, copperions, molybdenum ions, aluminium ions, iron ions, indium ions, galliumions, zirconium ions, and lanthanides. More preferred metal ions aremanganese ions, aluminium ions, tin ions and iron ions. Most preferredmetal ions are manganese ions.

Electrochemical Measurements

The present invention is not restricted to a particular electrochemicaltechnique. For example, potentiometry, controlled-current coulometry,amperometry, controlled-potential coulometry, stripping voltammetry,hydrodynamic voltammetry, polarography and stationary electrodevoltammetry, pulse polarography and voltammetry and cyclic voltammetrymay be used.

Herein, however, potentiometry is preferred, since it is simple,inexpensive and may be further miniaturized. Furthermore, potentiometryis a quantitative technique. The skilled person will readily understandthat and how the principle may be transferred to other electrochemicaltechniques.

The working electrode must be selected such that it responds to themetal ion used for binding and/or precipitation. This means that e.g.for a potentiometric detection the potential of the electrode depends onthe concentration of said metal ion. In certain cases it may beadvantageous that the electrode is selective for the metal ion. But thismay not be necessary in cases where the composition of the solution canbe controlled such as in the case of nucleic acid elongation. Preferredworking electrodes are electrodes based on manganese oxide. They show asignal which correlates to the concentration of the potentialdetermining ion, i.e. manganese, in solution.

As reference electrode, which provides a stable potential, for example asilver/silver chloride or a saturated calomel electrode (SCE) may beused in the inventive method. Preferably, the reference electrode is asilver/silver chloride electrode with a salt bridge. But alsominiaturized reference electrode constructions are possible.

In a specific embodiment, the metal ions are manganese ions and theelectrode is a manganese oxide electrode and the reference electrode isa silver/silver chloride electrode.

The present method has a number of advantages: The method is simple,cost-effective and may be easily implemented into existing devices.Moreover, since buffer conditions and components during pyrosequencingcan be highly controlled, even less specifically binding and/orprecipitating metal ions can be used to monitor the polymerase reaction.

Identification Procedure for Suitable Combination of Metal Ion/WorkingElectrode

In general, for identifying further working electrodes one may firstselect suitable metal ions and then chose the corresponding workingelectrode. The type of metal ion should be selected such that they meetone or more of the following criteria. Ideally, the metal ions selectedmeet all criteria: (1) The metal ions bind and/or precipitate PPi,preferably specifically. In some cases, metal ions binding to and/orprecipitating Phosphate (Pi) may be also used. In this case, apyrophosphatase should be added to the sample. (2) The metal ionsselected do not or not substantially bind to DNA, NTP, dNTP and proteinsor are efficiently blocked/displaced by other ions present in the buffersuch as magnesium. (3) The metal ions selected do not affect thechemical composition and structure of DNA and/or RNA. (4) The metal ionsselected do not affect (e.g. inhibit) the polymerase at typicalconcentrations used in the method. (5) The metal ions interact with theelectrode used in such a way that slight changes in the concentration orcomplex formation of metal ions can be measured.

By way of example, DNA is known to be damaged e.g. by platinum. Hence,such metals do not appear to be suitable. In contrast, tin can bepresent in at least two oxidation stated, i.e. Sn²⁺ and Se. It bindsmore specific to PPi (and Pi) than to ATP. It is assumed that tin doesnot bind strongly to DNA; it might, however, be necessary toadditionally add magnesium to the sample. Thus, tin may be analternative to manganese. Further, PPi (and/or Pi) binding metal ionsare molybdenum Mo, aluminium Al, iron Fe, gallium Ga, indium In,zirconium Zr and chromium Cr.

For identification of a suitable metal ion the candidate metal ionshould be assessed with regard to the foregoing criteria if not known.For example, Cigala et al. describe the interaction of Sn²⁺ and Zn²⁺with phosphate ligands (Cigala et al. 2012. J. Molecul. Liquids.165:143-153). Further, others describe the effect of different metalions on HIV-1 reverse transcriptase (Sabbioni et al. 1999. Biol. TraceElement Res. 68:107-119).

Electrochemical Nucleic Acid Sequencing Apparatus

The invention further relates to an electrochemical nucleic acidsequencing apparatus comprising: (a) means for carrying out the steps ofannealing and elongating a primer molecule to a template nucleic acid tobe sequenced; and (b) an electrochemical cell comprising metal ionsbeing capable of specifically binding to pyrophosphate and a workingelectrode responsive to said metal ions. In a preferred embodiment themetal ions are manganese ions and the electrode is based on manganesedioxide.

In a preferred arrangement these parts are combined in a small reactionchamber (see FIG. 2).

The means for carrying out the steps of annealing and elongating aprimer may comprise a temperature control unit for heating and cooling,such as one or more peltier elements. It may also comprise means forsupplying reagents and buffers to the reaction chamber. Such means cancomprise one or more channels (e.g. tubes). Preferably the reagents andbuffers can be supplied through different channels, i.e. one channel foreach nucleotide or alternatively for all nucleotides, one channel forthe sample, one channel for the polymerase, etc. The channels arepreferably controllable, e.g. by valves.

Example 1

Manganese ions and their depletion by enzymatically generatedpyrophosphate were followed by potentiometry using a manganeseoxide/carbon paste electrode. For this purpose a template DNA wasamplified by PCR using the following conditions:

Volume=50 μl

dNTP 10 mM 0.4 μl

Forward Primer 10 mM 0.2 μl Reverse Primer 10 mM 0.2 μl Template 2 μl 1u Taq Polymerase 10× Puffer (10 mM Tris, 50 mM KCl, 1.5 mM MgCl₂, pH 8.3(at 20° C.)) 5 μl MnCl₂ 10 mM 0.4 μl

Several samples from reaction mixtures after different numbers of PCRcycles yield different potentials (FIG. 1). It was shown that with asimple potential measurement the concentration of unbound Mn²⁺ ions canbe analyzed in a complex mixture and thus the progress in thebiochemical amplification reaction can be followed.

FIGURE CAPTIONS FIG. 1: MnO₂/Carbon-Paste-Electrode Measurement ofDifferent PCR Cycles.

Manganese ions and their depletion by enzymatically generatedpyrophosphate were followed by potentiometry using a manganesedioxide/carbon paste electrode. For this purpose a template DNA wasamplified by PCR using the conditions set out in the example section.

FIG. 2: Examples of the Electrochemical Sequencing Apparatus.

The upper part illustrates a measuring set up with a two electrodearrangement [metal ion sensitive working electrode (dark) and referenceelectrode (white)] dipping into the small reaction chambers andconnected to a voltmeter. The lower part shows schematically anintegrated version with enclosed working and reference electrodes withinthe reaction chambers.

1. An electrochemical nucleic acid sequencing method comprising thesteps of: a. providing a sample comprising a template nucleic acid to besequenced; b. annealing a primer molecule to said template nucleic acid,or creating a nick in the non-template strand for a site-specificelongation; c. carrying out nucleic acid elongation steps with apolymerase on said template in the presence of metal ions, wherein eachstep comprises the addition of a single type of nucleotide, whereinpyrophosphate is released in a stoichiometric ratio to the number ofnucleotides incorporated, and wherein the metal ions are capable ofpreferentially binding to pyrophosphate in solution; d. determining insaid sample the potential, current or charge at an electrode at leasttwice, wherein between the first time and the second time one or moreelongation steps take place or, alternatively, determining thepotential, current or charge continuously; and e. correlating the changein potential, current or charge over the number of elongation steps withthe type of nucleotides added.
 2. The method according to claim 1,wherein the nucleic acid sequencing method is based on pyrosequencing,reversible-terminator-pyrosequencing, or closed complex formationsequencing.
 3. The method according to claim 1 or 2, wherein step d. andoptionally step e. is done after each elongation step c.
 4. A method fordetecting a nucleic acid polymerase reaction comprising the steps of: a.providing a sample comprising a nucleic acid; b. carrying out a nucleicacid polymerase reaction step on said sample in the presence of metalions, wherein pyrophosphate is released or depleted depending on thetype of polymerase reaction, and wherein the metal ions are capable ofspecifically binding to pyrophosphate in solution; c. determining insaid sample the potential, current or charge at an electrode at leasttwice, wherein between the first time and the second time one or morenucleic acid polymerase reaction steps take place or, alternatively,determining the potential, current or charge continuously; and d.correlating the difference in potential, current or charge over thenumber of polymerase reaction steps and/or time with the progress of thenucleic acid polymerase reaction.
 5. The method according to claim 4,wherein the nucleic acid polymerase reaction is a synthesis reaction, anamplification reaction and/or a transcription reaction.
 6. The methodaccording to claim 4 or 5, wherein step c. and optionally step d. isdone after each polymerase reaction step b.
 7. A method of detecting theformation or depletion of pyrophosphate in a sample comprising the stepsof: a. providing a sample in which pyrophosphate is formed or depleted;b. adding to said sample metal ions, wherein the metal ions are capableof specifically binding pyrophosphate in solution; c. determining insaid sample the potential, current or charge at an electrode a first andat least a second time or continuously; d. correlating the difference inpotential, current or charge over time with the formation or depletionof pyrophosphate in the sample.
 8. A method of electrochemicallyquantifying pyrophosphate in a sample comprising the steps of: a. addingto a sample comprising pyrophosphate a predetermined amount of metalions or metal ions of a known potential, current or charge, wherein themetal ions are capable of specifically binding pyrophosphate insolution; b. determining in said sample the potential, current or chargeat an electrode; and c. correlating the potential, current or chargewith the amount of pyrophosphate present in said sample.
 9. The methodaccording to any of the preceding claims, wherein the metal ions areselected from the group of manganese ions, aluminium ions, tin ions andiron ions.
 10. The method according to any of the preceding claims,wherein the metal ions are manganese ions.
 11. The method according toany of the preceding claims, wherein the electrode is based on manganesedioxide.
 12. An electrochemical nucleic acid sequencing apparatuscomprising: a. means for carrying out the steps of annealing andelongating a primer molecule to a template nucleic acid to be sequenced;and b. an electrochemical cell comprising metal ions being capable ofpreferentially binding to pyrophosphate and at least one workingelectrode responsive to said metal ions.
 13. The electrochemical nucleicacid sequencing apparatus according to claim 12, wherein the metal ionsare manganese ions and the working electrode is based on manganesedioxide.